This invention relates to a process for production of crystals of L-methionine xcex3-lyase useful as an antitumor agent, a process for purification of L-methionine xcex3-lyase comprising the process for production of crystals and recombinant L-methionine xcex3-lyase crystals producable by the process for this producing system.
L-Methionine xcex3-lyase (EC 4.4.1.11) is an enzyme which requires pyridoxal phosphate as a coenzyme and catalyzes xcex1,xcex3-elimination and xcex3-replacement of L-methionine or its derivatives and also xcex1,xcex2-elimination and xcex2-replacement of S-substituted L-cysteine or its derivatives. It was reported that the enzyme was isolated and purified from Pseudomonas putida (Nakayama, T. et al., Anal. Biochem. 138, 421-424 (1984)). Recently, it was found that L-methionine xcex3-lyase has an antitumor activity (WO94/11535). In the past, L-methionine xcex3-lyase could be obtained in very small quantity from Pseudomonas. putida. However, recent development of recombinant DNA technology provides a possibility of its large quantity production (Inoue, H. et al., J. Biochem. 117, 1120-1125 (1995)).
Needless to say, a drug used as a pharmaceutical preparation should be pure. In the past, L-methionine xcex3-lyase was extracted from cells of Pseudomonas putida and purified by a combination of ion-exchange column chromatographies (Nakayama, T. et al., Anal. Biochem. 138, 421-424 (1984), Lishko, V K et al., Protein expression and purification 4, 529-533 (1993)). However, such enzyme did not have an enough purity to be used as pharmaceutical preparations, and it was difficult to purify it on a large scale.
As a method for production of protein crystals, a method using polyethylene glycol is well known. Concerning L-methionine xcex3-lyase, a procedure of its crystallization was reported (Esaki, N. et al., Methods in Enzymol. 143, 459-465 (1987)). The crystallization was performed by mixing L-methionine xcex3-lyase with a potassium phosphate buffer containing polyethylene glycol and leaving the mixture at room temperature (so called vapor diffusion method). In the paper, however, L-methionine xcex3-lyase of high purity which had previously been purified by column chromatographies was used for the crystallization. Moreover, the quantity of the crystals obtained was very small (1.6 mg).
It is difficult to produce a large quantity of crystals from a protein which has not been purified and contains impurities. Even though such crystallization succeeded, it was often impractical because the crystals still contain impurities. Therefore, a large scale crystallization at such step has hardly been attempted. This invention aims to provide a process for production of a large quantity of pure L-methionine xcex3-lyase crystals from unpurified L-methionine xcex3-lyase, and a process for purification of L-methionine xcex3-lyase which comprises the process for production.
From the result of intensive studies for the above purpose, the present inventors have found out that a highly purified L-methionine xcex3-lyase crystals can be produced in a short time by the method for production of L-methionine xcex3-lyase crystals using polyethylene glycol, that is to say, the first step is the warming of a solution containing L-methionine xcex3-lyase before or after addition of polyethylene glycol thereto and the second step is the addition of an inorganic salt. Thus, the present invention has been accomplished.
In this invention, L-methionine xcex3-lyase means either or both of a natural L-methionine xcex3-lyase produced by a microorganism such as Pseudomonas putida and a recombinant L-methionine xcex3-lyase prepared by a recombinant DNA technique. From the point of view for industrial mass production, the recombinant L-methionine xcex3-lyase is preferred. Therefore, though the recombinant L-methionine xcex3-lyase (hereinafter referred to as xe2x80x9crMETasexe2x80x9d) is used for explanations in the embodiments of the present invention, the natural L-methionine xcex3-lyase can also been used.
A solution containing L-methionine xcex3-lyase used in the process for producing the crystals of the invention means any of solutions which contain unpurified or purified L-methionine xcex3-lyase. Examples of the solution include a crude enzyme solution described in the following step 1, a solution containing unpurified L-methionine xcex3-lyase and a solution obtained after eliminating impurities by using polyethylene glycol. However, these examples do not limit the scope of this invention.
Additionally, cationic high molecular coagulants are used for eliminating nucleic acids or endotoxins as insoluble agglutinates by binding the cationic groups to anion groups of nucleic acids or endotoxins. Examples of the coagulants include polyethyleneimine or a cationic high molecular coagulant which mainly consists of chitosan (preferred is Kurimover I (Kurita Water Industries Ltd, Tokyo, Japan)). However, these examples do not limit the scope of this invention.